The importance of lipid membranes in the replication of RNA viruses is widely appreciated, yet our understanding of their precise chemical composition and function in this process remains poorly understood. While many studies have been able to document virus-induced changes in the expression of host enzymes responsible for lipid biosynthesis and metabolism, few studies have been able to translate these findings into chemical knowledge of the lipid species present in the membranes where RNA replication occurs, how the virus assembles this specialized membrane, how particular lipids dictate the biochemical and biophysical properties of these membranes, and how these properties in turn affect viral RNA replication. We discovered that hepatitis C virus (HCV) alters the abundance of desmosterol, a penultimate intermediate in cholesterol biosynthesis, and causes it to localize in the specialized membranes where RNA replication occurs. Depletion of desmosterol from the cell has a significant antiviral effect due to decreased steady-state RNA replication. Rescue of this effect by exogenous desmosterol cannot be fully recapitulated by cholesterol. Based on these findings, we have proposed a model in which desmosterol localized in the replication membrane is important for efficient RNA replication, and HCV perturbs the cholesterol biosynthetic pathway to promote accumulation of desmosterol at this locale. Here, we propose Aims designed to elucidate the unique mechanism(s) whereby HCV alters desmosterol homeostasis and to establish experimental systems that allow us to interrogate the effects of specific lipids on HCV RNA replication under chemically defined conditions. In Aim 1, we will probe the mechanisms by which HCV affects desmosterol homeostasis, focusing on HCV?s effects on DHCR24, the enzyme that converts desmosterol to cholesterol. We recently found that a post-translationally modified form of DHCR24 appears in HCV-infected cells and that the active NS3-4A protease is sufficient to generate this species. We have mapped the likely cleavage site near the N- terminus of the protein. Therefore, in Aim 1, we will examine whether this cleavage event affects the enzymatic activity, stability, or localization of DHCR24. In Aim 2, we will develop proteoliposome and supported lipid bilayer systems as models in which we can study the active HCV replicase within a membrane whose lipid content we can control and study. Since desmosterol differs from cholesterol only by the presence of an alkene at carbon 24, its distinct effects on HCV replication are an intriguing example that even seemingly subtle changes in lipid structure may have profound effects on membrane-associated biological processes. HCV?s mechanism for tuning lipid content in the membrane where replication occurs constitutes a novel class of virus-host interactions.